CN107674870A - A kind of method of the target RNA sequence of rna binding protein in improved identification of cell sample - Google Patents

A kind of method of the target RNA sequence of rna binding protein in improved identification of cell sample Download PDF

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CN107674870A
CN107674870A CN201610619492.1A CN201610619492A CN107674870A CN 107674870 A CN107674870 A CN 107674870A CN 201610619492 A CN201610619492 A CN 201610619492A CN 107674870 A CN107674870 A CN 107674870A
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张翼
薛亚强
张宇
陈栋
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ABLIFE (WUHAN) Inc
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Abstract

The present invention relates to biological technical field, there is provided a kind of identification of the improved target RNA sequence combined to intracellular rna associated proteins.Co-immunoprecipitation is crosslinked by UV and obtains the protein-bonded target RNAs of RNA, make not exclusively enzymolysis to the target RNA after UV-crosslinked using micrococcal nuclease, digest inactivates the enzyme after terminating using the chelating agent that can remove Ca2+ ions, the terminal phosphate of target RNA 3 ' and 3 ' RNA joints of connection that the above method is obtained, afterwards to connection product phosphorylation, separation and recovery, 5 ' RNA joints are connected afterwards, and RT PCR amplifications are carried out, obtain the corresponding cDNA libraries of target RNA.The present invention simplifies experiment flow, removes isotope marks step, adds IgG antibody negative control sample and deducts the influence of non-specific binding and background to experimental result, reaches the data result with Clip seq equal qualities.

Description

The target RNA sequence of rna binding protein in a kind of improved identification of cell sample Method
Technical field
The present invention relates to biological technical field, the target RNA sequence that more particularly to intracellular rna associated proteins are combined Identification.
Background technology
RNA is a kind of unstable large biological molecule, and most of RNA is required for and specific rna binding protein matter Combining to form RNA/ albumen compositions can just be stable in the presence of in cell；Moreover, it is dynamic between RNA and rna binding protein State association runs through and with the whole of RNA transcription synthesis, processing and modification, transmitter loss and positioning, Function and degraded Individual Life Cycles.
In consideration of it, separate or find that identification functional RNA molecule is one in RNA research fields using rna binding protein Indispensable research method.It is mainly by external index concentration aglucon to identify the conventional means of these target RNAs in the past Phyletic evolution technology (SELEX, Singh R, Valcarcel J, Green MR 1995Distinct binding specificities and functions of higher eukaryotic polypyrimidine tract-binding proteins.Science 268(5214)：1173-1176)；Co-immunoprecipitation obtains RNA- protein complexes in combination It is coupled technology (Buckanovich RJ, the Darnell RB 1997The neuronal RNA binding of gene microarray analysis protein Nova-1recognizes specific RNA targetsin vitro and in vivo.Mol.Cell.Biol.17(6)3194-3201；Darnell JC, Jensen KB, Jin P, Brown V, Warren ST, Darnell RB 2001Fragile X mental retardation protein targets G quartet mRNAs important for neuronal function.Cell 107 489-499；Brown V, Jin P, Ceman S, Darnell JC, O ' Donnell WT, Tenenbaum SA, Jin X, Feng Y, Wilkinson KD, Keene JD, Darnell RB, Warren ST 2001Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome Cell.107 (4)477-487).But the defects of very big be present in these means, cannot be distinguished by RNA and protein be direct interaction or Interaction is connect, and specific region for the RNA that protein is combined etc. can not be known.
RIP technologies (RNA Binding Protein Immunoprecipitation, rna binding protein immunoprecipitation), It is to study intracellular rna and the technology of protein binding situation, is the powerful for understanding post-transcriptional control network dynamic process. Howard Hughes Institute for Medical Research uses RIP technology combination high throughput sequencing technologies within 2010, i.e. RIP-seq technologies capture The RNAs that Polycomb complex proteins combine in full genome level, identifying PRC2 and more than 9000 RNAs has interaction pass It is (Jing Zhao, 1,2Toshiro K, Genome-wide Identification of Polycomb-Associated RNAs by RIP-seq.2010Molecμlar Cell；40,939–953).
Existing RIP-seq technical research object is all the interaction of RNA- protein weaker under nature in cell body Power, the target RNA molecule with protein interaction is easily lost in the operation of follow-up multi-step.Simultaneously because the target RNA molecule of capture Fragment is longer (being sequenced to obtain Insert Fragment with mRNA-seq in the same size), is unable to accurate identification RBP and RNA during subsequent analysis Binding site, many false positive findingses can be obtained, cause signal to noise ratio high (Zambelli＆Pavesi, 2015).
UV-crosslinked-Immunoprecipitation (Μ V Crosslinking-Immunopricipitation, CLIP) is A kind of experimental technique of the rise from 2003, (Μ is invented by the Darnell professors laboratory of Rockefeller University of the U.S. first Le J, Jensen KB, Ruggiu M, Mele A, Μ le A, Darnell RB 2003CLIP identifies NOVA- regulated RNA networks in the brain.Science 302(5648)1212-1215).The experimental technique knot The second generation high throughput sequencing technologies (The Second-Generation DNA Sequencing) risen recently are closed, are successfully reflected The motif of the target RNA of a variety of rna binding protein Binding in vivo and distribution (Yeo GW, Coufal in genome are determined NG, Liang TY, Peng GE, Fu XD, Gage FH 2009An RNA code for the FOX2splicing regulator revealed by mapping RNA-protein interactions in stem cells.Nat.Struct.Mol.)
Existing Clip-seq techniqueflows are complicated, and complex steps are, it is necessary to special instrument and equipment.Need to use and put simultaneously Penetrating property element32The position of the method indicator protein matter-RNA compounds of P marks, isotope have harm to human body, and country is to same Position element is strict using management, and common laboratory does not have qualification to go to apply for isotope.(Zambelli&Pavesi,2015)
The content of the invention
The present invention provides a kind of target RNA sequence of rna binding protein in improved identification of cell sample for above-mentioned deficiency The method of row, it comprises the following steps：
First, UV-crosslinked-co-immunoprecipitation obtains the target RNA of rna binding protein
1) UV-crosslinked and cell pyrolysis liquid preparation：
Using the ultraviolet light of 254m wavelength, 400mj/cm2Energy exposure cell, make direct interaction in cell Covalent bond is formed between protein and RNA, obtains protein-RNA complex；Wash is added into the cell precipitation of collection Buffer, obtain cell pyrolysis liquid；The addition RQ1DNA enzymes into cell pyrolysis liquid, TaKaRa RNase inhibitors, 1000rpm on the mixed instrument of Thermomixer, 37 DEG C, two minutes；Put 5 minutes on ice.It is then placed in the refrigerated centrifuge of 4 DEG C of precooling In 10,000rpm centrifuge 20 minutes, then with pipettor suction out supernatant.
Wash buffer：50ml 10X PBS, 0.5g SDS, 2.5g NaTDCs, 2.5ml NP-40, add ultra-pure water It is settled to 500ml.
2) fragmentation of RNA molecule：
Make not exclusively enzymolysis to the target RNA after UV-crosslinked using micrococcal nuclease MNase, make RNA tag at least More than 20 nucleotides, after enzymolysis terminates, the enzyme is inactivated using the chelating agent that can remove Ca2+ ions, obtains MNase Cell pyrolysis liquid after fragmentation
3) co-immunoprecipitation：
Albumin A or G magnetic beads are coupled respectively with the antibody and negative control IgG antibody for studying albumin X albumen respectively, 4 DEG C of one hours of mixing are put, after coupling, centrifuge tube is placed on Magneto separate frame, supernatant is suctioned out after magnetic bead precipitation；Add 1) magnetic bead is resuspended in the elution buffer in, puts to Magneto separate frame up to magnetic bead and precipitates, so in triplicate；MNase pieces in taking 2) Duan Huahou each 2 pipe of cell pyrolysis liquid, is added separately to X protein antibody-coupled magnetic beads and negative control IgG antibody coupled bead In, magnetic bead is resuspended until being all suspended in cell pyrolysis liquid, is placed on rotary mixer, puts 4 DEG C of one hours of mixing；With shifting Liquid device takes out supernatant；Wash buffer in being added 1) in combining the magnetic bead of research albumen and negative control IgG antibody, Lotion magnetic bead twice, adds High-salt wash buffer lotions magnetic bead twice as previously described, is finally buffered with 1 × PNK Liquid lotion magnetic bead is twice.
High-salt wash buffer：250ml10X PBS, 0.5g SDS, 2.5g NaTDCs, 2.5ml NP- 40, add ultra-pure water to be settled to 500ml；
1 × polynucleotide kinase buffer solution (1 × PNK b μ ffer)：25ml 1M Tris-HCl PH 7.4,1.0165g MgCl2, 2.5ml NP-40, add ultra-pure water to be settled to 500ml；
Elution buffer：5ml1M NaHCO3,0.5g SDS, add ultra-pure water to be settled to 50ml；
4) dephosphorylation：
X protein antibody magnetic bead and negative control IgG antibody magnetic bead are resuspended respectively with FAST AP reaction mixtures, is placed in 37 DEG C of 1000rpm concussions in every 3 minutes are incubated 10 minutes in 15 seconds on the mixed instrument of heat；Magnetic bead 2 is washed with 1ml 1 × PNK+EGTA buffer solutions It is secondary；Magnetic bead is washed with 1ml 1 × PNK buffer solutions 2 times；X protein and IgG magnetic beads is resuspended in Elution buffer respectively, is placed in heat 70 DEG C of 1000rpm are handled 10 minutes on mixed instrument, then are placed on Magneto separate frame, take supernatant to be transferred in new EP pipes after 1 minute；
Dephosphorylation mixed system (FAST AP reactions)：8 μ l 10X FAST AP buffer solutions, 4 μ l FAST AP, 68 μ l DEPC-H2O；
1 × PNK+EGTA buffer solutions：25ml1M Tris-HCl PH 7.4,3.8035g EGTA, 2.5ml NP-40, add Ultra-pure water is settled to 500ml；
5) RNA is extracted：
Elute solution and add Proteinase K, put on the mixed instrument of heat 55 DEG C, 1000rpm, 15s/3min are handled 2 hours；Trizol methods Extract RNA, DEPE-H2O back dissolvings；X protein and the RNA of IgG sample extractions carry out library construction；
6) 3 ' linker are connected：
Joint mixed system is added into 3 ' linker connection mixed systems, 22 DEG C is put in PCR instrument and reacts 2 hours；
Joint mixed system：5 μ l RNA, 1 μ l 3 ' linker (Seq ID NO:1), 70 DEG C of reaction 2min, put ice immediately On；
3 ' linker connection mixed systems：2 μ l 10X T4RNA ligase buffer solutions, 1 μ l T4RNA Ligase 1 (NEB), 1 μ l RRI, 3 μ l PEG8000,2 μ l 10mM ATP, 5 μ l DEPC-H2O；
7) 15% urea PAGE glue separates 3 ' linker connection products：
3 ' linker connection products are mixed with isometric RNA loading b μ ffer, 65 DEG C of heating 10min, then put In on ice, prepare loading after centrifugation；15% urea PAGE glue 180V electrophoresis 60min (bromophenol blue goes to blob of viscose bottom)；Electrophoresis knot Beam, under uviol lamp, in the blob of viscose that 39-54nt is cut on clean PE gloves.
15% urea PAGE glue formula：The acrylamide of 0.9ml 10xTBE, 3ml 45%, 5ml 7M urea liquids, 60 μ l 10%AP, 6 μ l TEMED；
8) electroelution recovery RNA：
9) phosphorylation：
Configure PNK phosphorylation systems：The linker connection products of 5 μ l 3 ', 1 μ l10X PNK B μ ffer, 1 μ l PNK, 1 μ l 10mM ATP, 0.5 μ l RRI, 1.5 μ l DEPC-H2O；Phosphorylation reaction mixed solution 37 DEG C, 30min, 70 DEG C in PCR instrument, 5min；
10) 5 ' linker are connected：
Take the linker of 1.0 μ l 5 ' (Seq ID NO:2), 70 DEG C, 2min, it is immediately placed on ice；Configure 5 ' linker connections Reaction system：10 μ l PNK Phosphorylated products, 1.5 μ l10X T4RNA ligase B μ ffer, 1 μ l5 ' linker, 0.15 μ l 100mM ATP, 1.3 μ l PEG8000,0.5 μ l RRI, 1 μ l T4RNA ligase 1 (NEB)；Reaction mixture is in PCR instrument 22 DEG C, 2 hours；
2nd, the target RNA for the rna binding protein that UV-crosslinked-co-immunoprecipitation obtains carries out cDNA synthesis
1) RT-PCR expands the DNA sequence dna of target RNA：
It is connected with to 15.45 μ l in 5 ' linker and 3 ' linker target RNA and adds the μ l of reverse transcriptase primer 1 (10 μM) (Seq ID NO:3), 65 DEG C of thermal shocks 5 minutes, are immediately placed on ice, moment quickly centrifuges；By 10 μ l reverse transcription reaction mixtures System and 15.45 μ l target RNAs and reverse transcriptase primer are mixed, and 50 DEG C of reaction 30min, 90 DEG C are reacted 5 minutes, 4 DEG C of insulation cDNA；Take 5 μ lcDNA enter performing PCR amplification as template, and reaction system is as follows:15.7μl DEPC-H2O,6μl5×HF Bμffer,1μ L10mM dNTP, 5 μ l cDNA, 1 10 μM of μ l PCR forward primers (Seq ID NO:4), 1 μ l, 10 μM of PCR reverse primers (Seq ID NO:3),0.3μl phusion hot start II DNA polymerase；Reaction condition is：98 DEG C be denatured 30 seconds, 98 DEG C denaturation 10 seconds, 60 DEG C take off fire 30 seconds, 72 DEG C extend 30 seconds, react 25-30 circulation, it is last 72 DEG C extension 5 minutes.Target RNA reverse transcription PCRs be DNA after, its size (length for including both ends linker) between 100-200bp, so on glue DNA in this section may be target sequence；
2) cDNA library reclaims:
12% PAGE glue electrophoresis, cuts the blob of viscose between 140-200bp；Electroelution reclaims, 10 μ l back dissolvings；illμ Mina Nex500 instrument high-flux sequences；High-flux sequence interpretation of result obtains Insert Fragment size and concentrates on 15-50bp, with IgG samples are background, carry out Peak Calling to X protein sample, obtain the special combination peak (peak) of experiment sample and go forward side by side Row statistics obtains the RNA labels of X protein in genome not same district with reference to the distribution situation of peak regional in reference gene group The distribution in domain；It is special to experiment sample with HOMER (Hypergeometric Optimization of Motif EnRichment) Different combination peak carries out motif analyses.
It is complicated, it is necessary to which specific apparatus is, it is necessary to use radioactive element for Clip-seq flows32P endangers asking for body Topic, the present invention simplify experiment flow, remove isotope marks step, add IgG antibody negative control sample and deduct non-specific knot Conjunction and influence of the background to experimental result, reach the data result with Clip-seq equal qualities.
Brief description of the drawings
Fig. 1 is existing CLIP-seq protocol procedures figure；
Fig. 2 is the present invention program flow chart；
Fig. 3 is the IP design sketch after certain albumen Μ V crosslinkings；
Fig. 4 is RNA fragment length statistical charts after the sequencing of library；
4A is patent flow library inserts size；4B is common RIP-seq library inserts size；
Fig. 5 is distribution maps of the reads in reference gene group after the sequencing of library；
5A represents patented method sequencing peaks and is distributed on genome；5B represents common RIP-seq and peaks is sequenced in base Because being distributed in group；
Fig. 6 is binding motifs displaying figure after the sequencing of library；
Embodiment
By combination accompanying drawing described further below it will be further appreciated that the features and advantages of the invention.The implementation provided Example is only explanation to the inventive method, remaining content without limiting the invention in any way announcement.
【Embodiment 1】RNA linker design and synthesis
The linker sequences used match with ill μm of ina Nex-500 sequenators：
3’linker：5‘-PO4ATC TGG AAT TCT CGG GTG CCA AGG-Puromycin 3’(Seq ID NO:1)；
5’linker：5‘Biotin-TGG AAT TCT CGG GTG CCA AGG 3’(Seq ID NO:2)；
The puromycin at 3 ' linker 3 ' ends and 5 ' linker 5 ' end biotins play closing linker ends and prevent head and the tail From effect even.
The reverse complemental DNA sequence dna that reverse transcriptase primer is 3 ' linker is i.e.：
5’CCT TGG CAC CCG AGA ATT C(Seq ID NO:3),
Forward primer used in PCR：
5’AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA(Seq ID NO:4)；
Reverse primer is reverse transcriptase primer.
【Embodiment 2】UV-crosslinked and cell pyrolysis liquid preparation
1) 150mm Tissue Culture Dish culture HeLa cells are used, culture medium treats Cell abundance with DMEM (Invitrogen) Culture medium is discarded when reaching more than 80%, after being rinsed 2 times with 1xPBS buffer solutions, appropriate PBS is added and covered cell Surface；
2) culture dish is placed in and on ice, be put into the drawer of HL-2000 crosslinkings instrument (Μ VP) bottom, make 400mj/cm2Energy The 254nm ultraviolets of amount irradiate 1 time.Culture dish is then taken out, removes PBS, 15ml PBS is added, is scraped with cell and scrape cell Under；
3) the PBS suspensions of all cells are sucked into 15ml centrifuge tubes with pipettor, 4 DEG C of 4000rpm are centrifuged 5 minutes, gone Clearly.Add 1.5ml PBS, blow afloat precipitation with pipettor, be transferred in clean 1.5ml centrifuge tubes (DEPC handle or use enter Mouth RNase free centrifuge tube), 4000rpm is centrifuged 5 minutes again, is removed supernatant, is put into -80 DEG C and saves backup or directly make With；
4) 600 μ l Wash buffer are added into the cell precipitation of collection, are blown and beaten several lower until cell precipitation is all heavy It is outstanding, 15-20 minutes on ice are placed in, cell is overturned every 5 minutes and mixed；
5) 30 μ l RQ1DNA enzymes (Promega) are added into cell pyrolysis liquid, 30 μ l RNase inhibitors (TaKaRa) are mixed Instrument (Thermomixer, Eppendorf) upper two minute (1000rpm, 37 DEG C)；
6) centrifuge tube is put 5 minutes on ice.It is then placed in 10,000rpm in the refrigerated centrifuge of 4 DEG C of precooling and centrifuges 20 points Clock, then supernatant is suctioned out with pipettor, retain 50 μ l and detected to Western.
Wash buffer：
【Embodiment 3】The fragmentation of RNA molecule
When first time carrying out RIP experiments to an albumen, it is necessary to grope most suitable micrococcal nuclease Micrococal N μ clease enzymes (MNase) (Fermentas companies) concentration, making RNA tag, at least in 20 nucleotides, (20 nucleotides are Μ CSC Genome Browser carry out genome alignment shortest length) more than, average length is advisable in 30-60 nucleotides.
1) Micrococal N μ clease enzymes are added according to the concentration groped, 10X MNase buffer solutions, is put in the mixed instrument of heat Upper 10 minute, 1000rpm, every 3 minutes mixed 15 seconds；
2) Ca2+ added in EDTA+EGTA solution chelating systems, terminating reaction.
【Embodiment 4】Co-immunoprecipitation
1) albumin A or G magnetic beads (Protein A beads, Dynal beads, Invitrogen 100.02) are taken respectively The antibody and negative control IgG antibody of 300 μ l and X protein are coupled respectively, put 4 DEG C of one hours of mixing, after coupling, Centrifuge tube is placed on Magneto separate frame, supernatant is suctioned out after magnetic bead precipitation；
2) 900 μ l Wash buffer (with embodiment 2) are added and magnetic bead are resuspended, put to Magneto separate frame up to magnetic bead and precipitate, So in triplicate；
3) each 2 pipes of the μ l of cell pyrolysis liquid 700 in Example 3 after MNase fragmentations, the X eggs in being added separately to 2) In white antibody-coupled magnetic beads and negative control IgG antibody coupled bead, magnetic bead is resuspended until being all suspended in cell pyrolysis liquid, It is placed on rotary mixer, puts 4 DEG C of one hours of mixing；
4) supernatant is taken out with pipettor, retains 50 μ l and detect IP efficiency to Western, remaining supernatant abandons；
5) 900 μ l Wash buffer are added in the magnetic bead of research albumen and negative control IgG antibody is combined, as before The lotion magnetic bead twice, adds High-salt wash buffer lotions magnetic bead twice, finally washed with 1 × PNK buffer solutions Agent magnetic bead is twice.(X antibody and the IP Efficiency testings of negative control IgG antibody are shown in Fig. 3)
High-salt wash buffer：
1 × polynucleotide kinase buffer solution (1 × PNK b μ ffer)：
【Embodiment 5】Dephosphorylation
1) dephosphorylation mixed system is configured：
2) X protein antibody magnetic bead and negative control IgG antibody magnetic bead are resuspended respectively with FAST AP reaction mixtures, put 10 minutes are incubated in 37 DEG C of 1000rpm concussions in every 3 minutes on the mixed instrument of heat within 15 seconds；
3) magnetic bead is washed 2 times with 1ml 1 × PNK+EGTA buffer solutions；
4) magnetic bead is washed 2 times with 1ml 1 × PNK buffer solutions；
5) X protein and IgG magnetic beads is resuspended in 100 μ l Elution buffer respectively, is placed in 70 DEG C of 1000rpm on the mixed instrument of heat Processing 10 minutes, then be placed on Magneto separate frame, take supernatant to be transferred in new EP pipes after 1 minute.
1×PNK+EGTA bμffer：
Elution buffer：
【Embodiment 6】Extract RNA
1) 100 μ l elution solution adds Proteinase K, puts on the mixed instrument of heat 55 DEG C, 1000rpm, 15s/3min processing 2 are small When；
2) extraction of Trizol methods RNA, 5 μ l DEPE-H2O back dissolvings.
【Embodiment 7】Connect 3 ' linker
X protein antibody capture RNA and negative control IgG antibody capture RNA build storehouse simultaneously.
1) joint mixed system is prepared：
70 DEG C of reaction 2min, put on ice immediately.
2) 3 ' linker connection mixed systems are prepared：
3) joint mixed system is added into 3 ' linker connection mixed systems, 22 DEG C is put in PCR instrument and reacts 2 hours.
【Embodiment 8】15% urea PAGE glue separates 3 ' linker connection products
1) 15% urea PAGE glue formula：
2) 3 ' linker connection products are mixed with isometric RNA loading buffer, 65 DEG C of heating 10min, then It is placed on ice, prepares loading after centrifugation；
3) 5 μ l 10bp DNA Ladder, 5 μ l oligo DNA (30bp, 40pmol/ μ l) are taken and in equal volume containing EB's RNA loading buffer are mixed, and prepare loading；
4) deposition condition：1xTBE, 180V prerunning 20min, loading (loading order from left to right：10bp DNA Ladder, oligo DNA, sample RNA, two holes are at least spaced between each sample), 180V electrophoresis 60min (go to by bromophenol blue Blob of viscose bottom)；
5) electrophoresis terminates, under uviol lamp, in the blob of viscose that 39-54nt is cut on clean PE gloves.
【Embodiment 9】Electroelution reclaims RNA
1) delivery port of electrophoresis tank is closed, pours into 0.5xTBE to putting glue groove top edge (can not have and, about 550ml)；
2) blob of viscose is put into and put in glue groove, write down the placement location of each sample, be carefully slowly added into V-groove 200 μ l 3M sodium acetates (pH5.2), 100V constant pressures elution 1h；
3) buffer solution in electrophoresis tank is slowly released to putting below glue groove breach from the delivery port of electrophoresis tank, it is careful to suction out Eluent in V-groove is into 1.5ml RNase-free EP pipes；
4) precipitation concentration tiny RNA after eluting：3 times of volume ethanols, -70 DEG C of 1-2h are added into eluent；
5) 13,200rpm, 4 DEG C of centrifugation 30min, abandon supernatant, the washing of the ethanol of 1m 75%, 13,200rpm, 4 DEG C of centrifugations 10min, supernatant, then brief centrifugation are abandoned, ethanol (careful operation, preventing RNA to be sucked away) are exhausted as far as possible with 200 μ l liquid-transfering gun, Appropriateness is dried, and adds 5 μ l DEPC-treated water to dissolve.
【Embodiment 10】Phosphorylation
1) PNK phosphorylation systems are configured：
2) phosphorylation reaction mixed solution is 37 DEG C, 30min, 70 DEG C in PCR instrument, 5min.
【Embodiment 11】Connect 5 ' linker
1) linker of 1.0 μ l 5 ' are taken, 70 DEG C, 2min, are immediately placed on ice；
2) 5 ' linker coupled reaction systems are configured：
3) mixed instrument is reacted 22 DEG C, 2 hours in PCR instrument.
【Embodiment 12】RT-PCR expands the DNA sequence dna of target RNA
1) be connected with to 15.45 μ l in 5 ' linker and 3 ' linker target RNA add it is inverse as described in example 1 above The μ l of transcription primers 1 (10 μM), 65 DEG C of thermal shocks 5 minutes, are immediately placed on ice, moment quickly centrifuges；
2) (reverse transcriptase SuperscriptIII and its buffer solution are purchased from preparation reverse transcription reaction mixed system Invitrogen companies)：
3) 10 μ l reverse transcription reactions mixed systems and 15.45 μ l target RNAs and reverse transcriptase primer are mixed, 50 DEG C of reactions 30min, 90 DEG C are reacted 5 minutes, 4 DEG C of insulation cDNA；
4) 5 μ lcDNA are taken to enter performing PCR amplification as template, reaction system is as follows:
Reaction condition is：98 DEG C are denatured 30 seconds, and 98 DEG C are denatured 10 seconds, and 60 DEG C take off fire 30 seconds, and 72 DEG C extend 30 seconds, reaction 25-30 circulation, last 72 DEG C extend 5 minutes.After target RNA reverse transcription PCR is DNA, its size (includes both ends linker's Length) between 100-200bp, so the DNA on glue in this section may be target sequence.
【Embodiment 13】CDNA library reclaims
1) 12% PAGE glue electrophoresis, cuts the blob of viscose between 140-200bp；
2) reclaimed with reference to the method electroelution of embodiment 9,10 μ l back dissolvings；
3) the illumina Nex500 instrument high-flux sequences of company.
4) high-flux sequence interpretation of result obtains Insert Fragment size and concentrates on 15-50bp (Fig. 4 A), and common RIP-seq But more than 140bp (Fig. 4 B) is concentrated on.IgG samples are background, carry out Peak Calling to X protein sample, are tested The special combination peak (peak) of sample simultaneously carries out distribution situation of the statistics with reference to peak regional in reference gene group, obtains X The RNA labels of albumen are in the distribution of genome different zones, as shown in Figure 5 5.75%, 3'UTR of 5'UTR distributions distributions 12.03%, CDS are distributed 54.85%, Introns and are distributed 11.94%, Intergenic distributions 0.36%.Use HOMER Combination peak (Hypergeometric Optimization of Motif EnRichment) special to experiment sample is carried out Motif is analyzed, and obtains before ranking as shown in Figure 63 Motif.

Claims (1)

1. a kind of method of the target RNA sequence of rna binding protein in improved identification of cell sample, it is characterised in that including Following steps：
First, UV-crosslinked-co-immunoprecipitation obtains the target RNA of rna binding protein
1) UV-crosslinked and cell pyrolysis liquid preparation：
Using the ultraviolet light of 254m wavelength, 400mj/cm2Energy exposure cell, make the albumen of the direct interaction in cell Covalent bond is formed between matter and RNA, obtains protein-RNA complex；Wash buffer are added into the cell precipitation of collection, are obtained Obtain cell pyrolysis liquid；RQ1 DNA enzymatics, TaKaRa RNase inhibitors are added into cell pyrolysis liquid, Thermomixer is mixed on instrument 1000rpm, 37 DEG C, two minutes；Put 5 minutes on ice；It is then placed in 10,000rpm centrifugations 20 in the refrigerated centrifuge of 4 DEG C of precooling Minute, then suction out supernatant with pipettor；
Wash buffer：50ml 10X PBS, 0.5g SDS, 2.5g NaTDCs, 2.5ml NP-40, add ultra-pure water constant volume To 500ml；
2) fragmentation of RNA molecule：
Make not exclusively enzymolysis to the target RNA after UV-crosslinked using micrococcal nuclease MNase, make RNA tag at least 20 More than individual nucleotides, after enzymolysis terminates, the enzyme is inactivated using the chelating agent that can remove Ca2+ ions, obtains MNase fragments Cell pyrolysis liquid after change；
3) co-immunoprecipitation：
Albumin A or G magnetic beads are coupled respectively with the antibody and negative control IgG antibody for studying albumin X albumen respectively, put 4 DEG C mixing one hour, after coupling, centrifuge tube is placed on Magneto separate frame, after magnetic bead precipitation after suction out supernatant；Add 1) In Wash buffer be resuspended magnetic bead, put to Magneto separate frame up to magnetic bead precipitate, so in triplicate；MNase fragments in taking 2) Each 2 pipe of cell pyrolysis liquid after change, is added separately in X protein antibody-coupled magnetic beads and negative control IgG antibody coupled bead, Magnetic bead is resuspended until being all suspended in cell pyrolysis liquid, is placed on rotary mixer, puts 4 DEG C of one hours of mixing；Use liquid relief Device takes out supernatant；Wash buffer in being added 1) in combining the magnetic bead of research albumen and negative control IgG antibody, such as The preceding lotion magnetic bead twice, adds high-salt buffer lotion magnetic bead twice, finally with 1 × PNK buffer wash magnetic bead two It is secondary；
High-salt wash buffer：250ml 10X PBS, 0.5g SDS, 2.5g NaTDCs, 2.5mlNP-40, add Ultra-pure water is settled to 500ml；
1 × polynucleotide kinase buffer solution (1 × PNK b μ ffer)：25ml 1M Tris-HCl PH 7.4,1.0165g MgCl2, 2.5ml NP-40, add ultra-pure water to be settled to 500ml；
Elution buffer：5ml 1M NaHCO3,0.5g SDS, add ultra-pure water to be settled to 50ml；
4) dephosphorylation：
X protein antibody magnetic bead and negative control IgG antibody magnetic bead are resuspended respectively with FAST AP reaction mixtures, it is mixed to be placed in heat 37 DEG C of 1000rpm concussions in every 3 minutes are incubated 10 minutes in 15 seconds on instrument；Magnetic bead is washed with 1ml 1 × PNK+EGTA buffer solutions 2 times； Magnetic bead is washed with 1ml 1 × PNK buffer solutions 2 times；X protein and IgG magnetic beads is resuspended in Elution buffer respectively, is placed in the mixed instrument of heat Upper 70 DEG C of 1000rpm are handled 10 minutes, then are placed on Magneto separate frame, take supernatant to be transferred in new EP pipes after 1 minute；
Dephosphorylation mixed system (FAST AP reactions)：8 μ l 10X FAST AP buffer solutions, 4 μ l FASTAP, 68 μ l DEPC- H2O；
1 × PNK+EGTA buffer solutions：25ml1M Tris-HCl PH 7.4,3.8035g EGTA, 2.5mlNP-40, add ultra-pure water It is settled to 500ml；
5) RNA is extracted：
Elute solution and add Proteinase K, put on the mixed instrument of heat 55 DEG C, 1000rpm, 15s/3min are handled 2 hours；Trizol methods are extracted RNA, DEPE-H2O back dissolvings；X protein and the RNA of IgG sample extractions carry out library construction；
6) 3 ' linker are connected：
Joint mixed system is added into 3 ' linker connection mixed systems, 22 DEG C is put in PCR instrument and reacts 2 hours；
Joint mixed system：5 μ l RNA, 1 μ l 3 ' linker (Seq ID NO:1), 70 DEG C of reaction 2min, put on ice immediately；
3 ' linker connection mixed systems：2 μ l 10X T4RNA ligase buffer solutions, 1 μ l T4RNA Ligase1 (NEB), 1 μ l RRI, 3 μ l PEG8000,2 μ l 10mM ATP, 5 μ l DEPC-H2O；
7) 15% urea PAGE glue separates 3 ' linker connection products：
3 ' linker connection products are mixed with isometric RNA loading b μ ffer, 65 DEG C of heating 10min, are subsequently placed in ice On, prepare loading after centrifugation；15% urea PAGE glue 180V electrophoresis 60min (bromophenol blue goes to blob of viscose bottom)；Electrophoresis terminates, Under uviol lamp, in the blob of viscose that 39-54nt is cut on clean PE gloves；
15% urea PAGE glue formula：The acrylamide of 0.9ml 10xTBE, 3ml 45%, 5ml 7M urea liquids, 60 μ l 10% AP, 6 μ l TEMED；
8) electroelution recovery RNA：
9) phosphorylation：
Configure PNK phosphorylation systems：The linker connection products of 5 μ l 3 ', 1 μ l10X PNK B μ ffer, 1 μ l PNK, 1 μ l 10mM ATP, 0.5 μ l RRI, 1.5 μ l DEPC-H2O；Phosphorylation reaction mixed solution is 37 DEG C, 30min, 70 DEG C in PCR instrument, 5min；
10) 5 ' linker are connected：
Take the linker of 1.0 μ l 5 ' (Seq ID NO:2), 70 DEG C, 2min, it is immediately placed on ice；Configure 5 ' linker coupled reactions System：10 μ l PNK Phosphorylated products, 1.5 μ l10X T4RNA ligase B μ ffer, 1 μ l5 ' linker, 0.15 μ l 100mM ATP, 1.3 μ l PEG8000,0.5 μ l RRI, 1 μ l T4RNA ligase 1 (NEB)；Reaction mixture is 22 DEG C in PCR instrument, and 2 Hour；
2nd, the target RNA for the rna binding protein that UV-crosslinked-co-immunoprecipitation obtains carries out cDNA synthesis
1) RT-PCR expands the DNA sequence dna of target RNA：
Addition reverse transcriptase primer 1 μ l (10 μM) (Seq in 5 ' linker and 3 ' linker target RNA is connected with to 15.45 μ l ID NO:3), 65 DEG C of thermal shocks 5 minutes, are immediately placed on ice, moment quickly centrifuges；By 10 μ l reverse transcription reactions mixed systems and 15.45 μ l target RNAs and reverse transcriptase primer are mixed, and 50 DEG C of reaction 30min, 90 DEG C are reacted 5 minutes, 4 DEG C of insulation cDNA；Take 5 μ LcDNA enters performing PCR amplification as template, and reaction system is as follows:15.7μl DEPC-H2O,6μl5×HF Bμffer,1μl10mM DNTP, 5 μ l cDNA, 1 μ l10 μM PCR forward primers (Seq ID NO:4), 1 μ l, 10 μM of PCR reverse primers (Seq ID NO: 3),0.3μl phusion hot start II DNA polymerase；Reaction condition is：98 DEG C are denatured 30 seconds, 98 DEG C of denaturation 10 seconds, 60 DEG C took off fire 30 seconds, and 72 DEG C extend 30 seconds, reacted 25-30 circulation, and last 72 DEG C extend 5 minutes.Target RNA reverses PCR is recorded as after DNA, its size (length for including both ends linker) is between 100-200bp, so on glue in this section DNA may be target sequence；
2) cDNA library reclaims:
12% PAGE glue electrophoresis, cuts the blob of viscose between 140-200bp；Electroelution reclaims, 10 μ l back dissolvings；illμmina Nex500 instrument high-flux sequences；High-flux sequence interpretation of result obtains Insert Fragment size and concentrates on 15-50bp, with IgG samples This is background, carries out Peak Calling to X protein sample, obtains the special combination peak (peak) of experiment sample and counted With reference to the distribution situation of peak regional in reference gene group, point of the RNA labels in genome different zones of X protein is obtained Cloth；With HOMER (the Hypergeometric Optimization of Motif EnRichment) knots special to experiment sample Close peak and carry out motif analyses.
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CN111909991B (en) * 2019-05-09 2021-08-03 中国科学院生物物理研究所 Method for capturing RNA in-situ high-grade structure and interaction
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CN111020018A (en) * 2019-11-28 2020-04-17 天津金匙医学科技有限公司 Macrogenomics-based pathogenic microorganism detection method and kit
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